Preparative Denaturing Isoelectric Focusing for Enhancing Sensitivity of Proteomic Studies

Abstract

Substantial evidence implicates important roles for both protein phosphorylation and protein degradation in regulation of plant defense responses. Therefore, interest is growing in applying proteomics techniques to investigate these posttranscriptional changes. We have found, however, that most proteins of interest are not visible on two-dimensional (2D) gels without previous prefractionation. This chapter describes the use of preparative denaturing isoelectric focusing to enrich for proteins of specific isoelectric points before separation by 2D gels. This method significantly increases the sensitivity of 2D gelbased comparisons.

1 Introduction

After recognition of microbial elicitors, plants initiate a number of rapid defense responses. Although it is clear that phosphorylation plays a key role in initiating these responses, little is known about the regulatory proteins involved. Although it is possible to use radioactive pulse-labeling of phosphoproteins in cell culture to identify signaling components (1,2), we have found that the majority of radioactively labeled proteins cannot be aligned with stained protein spots on two-dimensional (2D) gels using total protein extracts. Signaling proteins are typically low in abundance, and phosphorylation is rarely an event of high stoichiometry. This combination of factors generally means that the target, modified forms of the proteins will be present at exceedingly low levels. Because there is a restriction in the amount of total protein that can be loaded onto 2D gels, simply increasing the load is not an option. Thus, prefractionation of protein samples is essential for meaningful proteomic comparisons. We have found preparative liquid isoelectric focusing (IEF; e.g. Rotofor IEF, Bio-Rad) a powerful and complementary tool for the identification of proteins using 2D gels and mass spectrometry. This procedure prefractionates proteins on the same principles as the first-dimension IEF separation for 2D gels. The end result is that rather than loading 1 mg of total protein on a single isoelectric point (pI) unit zoom gel, resulting in the majority of protein focusing outside of the target region, IEF prefractionation allows loads of 1 mg of protein only from the pI unit of interest. Thus, this method substantially increases sensitivity and allows identification of even rare proteins.

2 Materials

• 1. Protein extraction buffer: 100 mM HEPES-KOH, pH 7.5, 5% glycerol, 50 mM sodium pyrophosphate, 1 mM sodium molybdenate, 25 mM sodium fluoride, 15 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid, 5 mM ethylene diamine tetraacetic acid (EDTA), 0.5% polyvinylpyrrolidine, 1% triton (3 mM dithiothreitol added on day of use).

• 2. 100 mM Phenylmethyl sulfonyl fluoride (PMSF) in isopropanol (store at 4°C).

• 3. 10 mM Leupeptin (store at −20°C).

• 4. 10 µM Calyculin A (store at −20°C).

• 5. Phenol.

• 6. Back extraction buffer: 100 mM Tris-HCl, pH 8.4, 20 mM KCl, 10 mM EDTA, 0.4% β-mercaptoethanol.

• 7. 100 mM Ammonium acetate in methanol.

• 8. 80% Acetone, 50 mM Tris-HCl, pH 8.0.

• 9. Sonicating water bath.

• 10. Low-stringency buffer: 9 M urea, 1% Triton X-100, 5% ampholytes, 0.5% DTT.

• 11. High-stringency buffer: 7 M urea, 2 M thiourea, 4% CHAPS, 5% ampholytes, 2% DTT.

• 12. 50% Glycerol.

3 Methods

3.1 Initial Protein Extraction

This protocol is a modification of that previously described by Peck et al. (2) and yields proteins of good quality for IEF separation. Attempts to perform IEF with samples of insufficient purity (i.e., directly using a protein extract) will generally result in poor focusing.

• 1. Immediately before use, add inhibitors to protein extraction buffer (final concentrations of 1 mM PMSF, 10 µM leupeptin, and 10 nM Calyculin A; see Note 1). We recommend using approx 2 mL extraction buffer per gram of fresh weight.

• 2. Centrifuge (10,000g, 10 min) to clear cell debris.

• 3. Transfer the supernatant to a tube containing 1 vol of phenol, vortex, and keep on ice for 5 min.

• 4. Centrifuge (10,000g, 10 min) and discard the aqueous phase. Be careful not to disturb the interface (protein will be in the phenol phase and interface).

• 5. Add one volume of back extraction buffer, vortex, and centrifuge (10,000g, 10 min). Discard the aqueous phase, and repeat the back extraction. Steps 3 to 5 will help remove both nucleic acids and sugars that would interfere with protein focusing.

• 6. Add 5 vol of 100 mM ammonium acetate in methanol, vortex, and place at −20°C for 20 min to precipitate proteins from the phenol.

• 7. Centrifuge (10,000g, 10 min) to pellet protein.

• 8. Wash the pellet with 100 mM ammonium acetate in methanol (see Note 2), using a sonicating water bath to break up the pellet. Centrifuge (10,000g, 10 min) and repeat wash.

• 9. Wash the protein pellet from previous step with 80% acetone buffered with 50 mM Tris-HCl, pH 8.0. Again, use a sonicating water bath to break up the pellet. Centrifuge (10,000g, 10 min) and repeat wash. Protein can be stored as an acetone suspension indefinitely at −20°C.

3.2 Preparative Isoelectric Focusing

• 1. After thoroughly mixing the acetone suspension, remove an amount equivalent to 20 to 40 mg of protein to a fresh tube (see Note 3). Centrifuge (10,000g, 10 min), discard supernatant, and allow pellet to air-dry.

• 2. Resuspend pellet in denaturing IEF buffer (see Note 4). Determine the volume capacity for the IEF unit (see manufacturers instructions). Prepare the proper amount of chemicals for half this volume to be prepared with double-distilled water and the other half to be prepared with 50% glycerol. The glycerol is necessary to prevent protein precipitation during focusing, but the proteins will dissolve more easily in the glycerol-free buffer.

• 3. Resupend the pellet in the buffer without glycerol for 1 h with continuous shaking. It may be necessary to assist resuspension by pipetting and/or using the sonicating water bath.

• 4. Centrifuge (10,000g, 10 min) to remove unsolubilized material.

• 5. Mix the supernatant with the glycerol-containing buffer. The proteins are ready to be loaded in the IEF cell.

• 6. Follow manufacturers instructions for assembling the IEF unit, loading the sample, and focusing conditions. (Specifics may vary depending on IEF unit.)

• 7. After focusing, you will have many (approx 20) fractions representing a range of pIs ( Fig. 1 ). We have generally found that mixing two to three adjacent fractions does not greatly decrease sensitivity and substantially reduces the number of samples for processing in step 8.

• 8. Fractions need to be back-extracted to remove ampholytes that would interfere with the ampholyte composition of the IEF strip for first dimension separation of 2D gels. Perform steps 3–9 from Subheading 3.1. on each sample.

• 9. Again, samples can be stored as acetone suspensions until needed.

• 10. To use the appropriate first dimension pH gradient for each fraction, we perform an immunoblot analysis using a protein of known pI (see Fig. 2 ).

• 11. Alternatively, pH 3.0–10.0 linear-gradient 2D gels can be run with a small amount of sample to determine the corresponding gradients for each fraction.

Fig. 1.

Fractionation of proteins by preparative isoelectric focusing. (A) Protein samples resuspended in a denaturing buffer, mixed with ampholytes, and separated by isoelectric focusing. When focusing is complete, fractions are vacuum collected for further analysis. (B) Separation of protein samples (20 µg) from preparative isoelectric focusing fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis show changes in protein patterns between fractions.

Fig. 2.

Using immunoblot analysis to determine isoelectric point (pI) cut-off from preparative isoelectric focusing fractions. After preparative isoelectric focusing of proteins from Lotus japonicus, sequential fractions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride. Immunoblots were hybridized with an antibody raised against Phos43 (2), which has a pI of less than 4.3. As seen in the immunoblot analysis, proteins cross-reacting with the Phos43 antibody are found only in the most acidic fractions, indicating that these fractions can be run on expanded pI gradients with pIs less than 4.5.

4 Notes

• 1. The protein extraction buffer given is for isolation of phosphoproteins. If phosphorylation status is not a consideration, phosphatase inhibitors can be eliminated (i.e., sodium pyrophosphate, sodium molybdenate, sodium fluoride, and Calyculin A). PMSF and Calyculin A are unstable in solution, so they should be added only immediately before protein isolation. The polyvinylpyrrolidine (PVP) is present to bind and remove polyphenolics that might otherwise damage proteins. For older leaves or leaves from difficult species (e.g., solanaceae), it may be necessary to increase the PVP concentration or to include the insoluble polyvinylpolypyrrolidine to remove all polyphenolics. A simple indication is that if the protein extract is turning purple, more PVP or polyvinylpolypyrrolidine should be used.

• 2. After the first methanol precipitation, the protein pellet tends to be spread along the entire wall of the centrifuge tube. Be sure to use a pipet to remove all protein from the wall of the tube with the first wash. Failure to do so will result in significant losses of protein. This step is generally not necessary after subsequent centrifugations.

• 3. We generally use protein concentrations of approx 0.5 mg/mL. Higher protein concentrations are possible but could result in protein precipitation because of the desalting effect of focusing or the increase of local protein concentration. It may be possible to overcome these problems by increasing ampholyte concentrations (we use 5%, but they theoretically can be raised up to 40%) or by adding more glycerol to the buffer (we use 12.5%, but it may be possible to increase the concentration to 20%).

• 4. We sometimes find it advantageous to decrease the complexity of the proteome using differential protein extraction from the acetone pellet. The low-stringency buffer resolubilizes a subset of proteins with a general bias toward smaller (<60 kDa) proteins. The remaining pellet can then be resuspended with the highstringency buffer. In both cases, the amount of urea and/or thiourea is substantial and affects the volume of the solution. Therefore, only add approx 2.9 mL of water for each 5 mL of total volume of low-stringency buffer or 2.5 mL of water for each 5 mL of the high-stringency buffer. Once the chemicals are fully resuspended, adjust the final volume. For the solutions containing glycerol, perform the same procedure but use 50% glycerol instead of water to make the buffers.